According to the World Health Organization, about 10 million cigarettes are sold every minute in the world and every eight seconds someone dies from tobacco use (WHO, 2002). Nicotine is the main addictive component of tobacco, and the affective and somatic symptoms associated with nicotine withdrawal contribute to the difficulty in quitting tobacco use. While current therapies for smoking cessation are helpful, none can claim a very high rate of success. Therefore, there is a great need for a better understanding of the behavioral and biological factors underlying nicotine withdrawal. The persistence of cigarette smoking is influenced by genetic factors as highlighted by the recently found correlation between nicotine dependence and single nucleotide polymorphism (SNP) in the CHRNA5-CHRNA3-CHRNB4 nAChR gene cluster. This application builds on the observation that lack of ?5-containing nicotinic acetylcholine receptors (?5* nAChRs) abolishes somatic signs of withdrawal and reduces anxiety-like responses. ?5* nAChRs are expressed in the medial habenula (MHb) and interpeduncular nucleus (IPN). Recent studies from the lab have shown that injection of the nAChR antagonist, mecamylamine, in either the MHb or IPN is sufficient to precipitate withdrawal signs in nicotine-treated mice. We will first ask whether the rs16969968 SNP, which causes a D398N aminoacid change, can alter the biophysical and pharmacological properties of the ?5* nAChR subtypes expressed in MHb and IPN. Patch clamp experiments will be conducted in heterologous expression systems on nAChRs comprising the D398 or N398 ?5 subunit variants. Second, to investigate the direct role of ?5* nAChRs in MHb and IPN, lentiviral vectors will be used to either express ?5 gene variants in the MHb and IPN of ?5 null mice or to knock down the levels of endogenous ?5 in the MHb and IPN of wild type mice. MHb and IPN slices from those mice will be used in patch clamping experiments to determine the effect of the genetic manipulations on nicotine-induced increases in spike firing frequency. In other experiments, lentivirus-injected mice will be exposed to nicotine in the drinking water for six weeks to study mecamylamine-precipitated withdrawal. The studies will determine whether expression of ?5* nAChRs is necessary and sufficient for the manifestation of nicotine withdrawal and whether the potential molecular changes produced by the rs16969968 15 SNP translate into different withdrawal symptoms in vivo. PUBLIC HEALTH RELEVANCE: Our studies will take advantage of genetically engineered mice and novel genetic techniques, and will combine behavioral analysis, molecular biology, and pharmacology to study the influence of an identified single nucleotide polymorphism on the manifestations of nicotine withdrawal. The studies could lead to the development of personalized smoking cessation therapies.